An efficient molecular design breeding strategy for grape coloring - PowerPoint PPT Presentation

An efficient molecular design breeding strategy for grape coloring trait based on MYB haplotypes Le Guan, Songtao Jiu, Haifeng Jia and Jinggui Fang * College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, P.R. China Email: fanggg@njau.edu.cn XII International Conference on Grapevine Breeding and Genetics 15-20, July 2018. Bordeaux, France

Grapevine is one of the most important fruit trees in China Grapevine cultivation in China  Wide distribution  High yields  High economic benefits  Mainly consumed as fresh fruits

Anthocyanin composition and color diversity Anthocyanin composition is responsible for the color diversity of grape berries.

The MYB haplotype is the genetic determinant of grape color Alleles Chr2 VvmybA1a Color locus VvmybA1b MybA1 VvmybA1c MybA2 VvmybA1 SUB VvmybA1 BEN MybA1 and MybA2 , VlmybA1-3* inherited together, can be regarded as the color locus . Gret1 : grapevine retrotransposon1 LTR: long terminal repeat TS: target site for the Gret1 insertion Alleles VvmybA2w VvmybA2r VlmybA1-2* VlmybA2* (Kobayashi et al. 2004, Walker et al. 2006, Azuma et al. 2009, 2011)

Haplotype composition at the color locus is a major genetic determinant of skin color A model of the evolutionary differentiation of MYB haplotypes at the color locus in Vitis species (Azuma et al. 2017)

Our goals and questions raised Assisting in breeding of high quality cultivars with favorable coloration Can we achieve early prediction of color diversification according to the MYB haplotype composition?

Coloration classification of the 213 investigated grape varieties Numbers of grape varieties The 213 grape varieties were classified into 7 categories according to the depth of the coloration.

Identification of MYB haplotype composition of the 213 grape varieties by PCR and sequencing 1675 bp VvMybA1b VvMybA1a 1559 bp 846 bp 2105 bp VvMybA1 BEN VvMybA1c 1035 bp + VvMybA1 SUB sequencing 999 bp + VlMybA1-3 sequencing MybA1 allele identification DNA quality control by the amplification of actin

Identification of MYB haplotype composition of the 213 grape varieties by PCR and sequencing 1446 bp + VvMybA2r sequencing VlMybA2 161 bp 1444 bp + VvMybA2w sequencing 251 bp VlMybA1-2 DNA quality control by the amplification of actin MybA2 allele identification

MYB haplotype composition of the investigated grape varieties G/F G/C-N F V. vinifera x V. labrusca C-Rs/E1/E2 V. vinifera C-Rs C-N/E2 C-N A/F A/E2 A/E1/E2 A/E1 A/C-Rs/E2 A/C-Rs/E1 A/C-Rs A/C-N A/B MYB haplotype composition of 211 grape varieties can be identified by PCR and sequencing except ‘ Yuanruihei ’ ( V. vinifera ) and ‘ Olarra Queen’ ( V. labrusca ) .

MYB haplotype composition of ‘Yuanruihei’ and ‘Olarra Queen’ HapA ‘phased phase’ HapC-N MYB haplotype composition of ‘Yuanruihei’ and ‘Olarra Queen’ could be HapC-Rs  either A/C-N or G/C-Rs ‘repulsive phase’ HapG  identified by self-crossing

Identification of MYB haplotype composition of ‘Yuanruihei’ Haplotype VvMybA1 SUB VvMybA1 BEN VvMybA2r/w Code VvMybA1a VvMybA1b VvMybA1c composition 1 + 2w A 2 + 2w A 3 + 2w A 4 + 2w A 5 + 2w A 6 + 2w A 7 + 2w A 8 + 2w A 9 + 2w A 10 + + 2w/2r AC-N 11 + + 2w/2r AC-N 12 + + 2w/2r AC-N 13 + + 2w/2r AC-N 14 + + 2w/2r AC-N 15 + + 2w/2r AC-N 16 + + 2w/2r AC-N 17 + + 2w/2r AC-N 18 + + 2w/2r AC-N 19 + + 2w/2r AC-N 20 + + 2w/2r AC-N 21 + + 2w/2r AC-N 22 + + 2w/2r AC-N 23 + + 2w/2r AC-N 24 + + 2w/2r AC-N 25 + + 2w/2r AC-N 26 + 2r C-N 27 + 2r C-N Haplotype composition of 28 + 2r C-N 29 + 2r C-N ‘ Yuanruihei ’ was A/C-N. 30 + 2r C-N 31 + 2r C-N 32 + 2r C-N 33 + 2r C-N

Identification of MYB haplotype composition of ‘ Olarra Queen’ Haplotype VvMybA1 SUB VvMybA1 BEN VvMybA2r/w Code VvMybA1a VvMybA1b VvMybA1c composition 1 + 2w A 2 + 2w A 3 + 2w A 4 + 2w A 5 + 2w A 6 + 2w A 7 + + 2w/2r AC-N 8 + + 2w/2r AC-N 9 + + 2w/2r AC-N 10 + + 2w/2r AC-N 11 + + 2w/2r AC-N 12 + + 2w/2r AC-N 13 + + 2w/2r AC-N 14 + + 2w/2r AC-N 15 + + 2w/2r AC-N 16 + + 2w/2r AC-N 17 + + 2w/2r AC-N 18 + + 2w/2r AC-N 19 + 2r C-N 20 + 2r C-N 21 + 2r C-N 22 + 2r C-N 23 + 2r C-N 24 + 2r C-N Haplotype composition of 25 + 2r C-N ‘ Yuanruihei ’ was A/C-N.

Is coloration and haplotype composition closely correlated? V. vinifera V. vinifera x V. labrusca  A total of 8 haplotypes and 19 haplotype compositions were identified;  HapC-N and HapE2 showed stronger effect than HapC-Rs, HapB and HapE1;  And thus VlmybA2 might have stronger effect than Vlmyb1-2 regarding coloration;  The more functional alleles, the darker the skin tended to be.

Can we achieve early color prediction during cross breeding? a) Haplotype identification b) The berry color observation and berry coloration and quantification ‘Muscat Hamberg’ ♀ ‘Crimson seedless’ ♂ AC-Rs AC-Rs × : : F1 A/A A/C-Rs C-Rs/C-Rs 1 Expected 1 2 19 Observed a 18 32 Berry color < < a Chi square value=0.391, P value=0.82

Can we achieve early color prediction during cross breeding? a) Haplotype identification and b) The berry color observation berry coloration and quantification ‘Cuibao seedless’ ♀ ‘Qiuhongbao’ ♂ A/A A/C-Rs × : A/A F1 A/C-Rs Expected 1 1 Observed a 38 25 Berry color a Chi square value=2.29, P value=0.13

Summary ‘Muscat Hamberg’ × ‘Crimson seedless’ Cuibao seedless’ × ‘Qiuhongbao’ Confirmed Verification  The berry coloration matched with the haplotype Early prediction of color composition in 99.1% of the investigated varieties; diversification can be achieved  AC-N and AE2 tended to show deeper colors; by the identification of the MYB  The more functional alleles it contained, the haplotype composition darker the skin color tended to be Closely correlated An efficient molecular design MYB haplotype breeding strategy for grape coloring Color index composition trait based on MYB haplotypes 213 grape varieties

Potential strategy for the way forward Selection of multiple candidate parents with favorable target traits from the germplasm Multiple target breeding traits MYB haplotype database cross-check Preference parents with MYB haplotype generating target color trait x More hybrids with desirable target traits including berry color

Acknowledgements Fundings Zhengzhou Fruit Research Institute, CAAS National Natural Science Foundation of China Ministry of Technology and Science, P.R. China Thank you for your attention!